Abstract Historically, treatment options for melanoma were limited, and 5-year survival rates were <10% for patients with advanced-stage disease. Recently, immune checkpoint inhibitors (ICIs) have shown encouraging success for treatment of melanoma. However, approximately 40% of patients with advanced melanoma have primary ICI resistance and sub-populations of initially responding patients develop secondary resistance. Can we identify druggable molecular pathways in these non-responders to increase their ICI responsiveness and potentially save lives? We have performed the only reported proteomics investigation of pre-ICI treatment melanoma biopsies from responders and non-responders (6). In this preliminary study, we were able to identify a panel of protein and histone epigenetic signatures that provided evidence for dysregulated molecular pathways correlated to ICI responsiveness ? providing the scientific premise for the outlined studies. This preliminary study will be expanded to include 1) a larger number of patients, 2) patients with primary and secondary ICI therapy resistance, and 3) tumor cell-type analyses. The overall goal of this proposal is to combine state-of-the-art proteomics of patient tissues with pre-clinical animal models of ICI responsiveness to create a functional proteomics pipeline for uncovering cellular protein and histone epigenetic pathways functionally dysregulated in melanoma ICI non-responders. We will use a rigorous strategy to target gene transcription and histone modifying proteins as these are upstream pathway effectors that can be druggable. We hypothesize that certain proteins involved in gene transcription and posttranslational modification of histones will be dysregulated in ICI non-responders and functionally coupled to molecular mechanisms of ICI responsiveness, which will identify these proteins as high-priority, druggable candidates for complementing existing ICI therapy. The aims of this study are: 1) perform functional proteomics to identify dysregulated gene transcription protein pathways in melanoma ICI non-responders, 2) use high-resolution mass spectrometry to identify dysregulated histone modifying protein pathways in melanoma ICI non-responders, and 3) determine the functional significance in antitumor responses to ICI therapy for gene transcription and histone modifying proteins dysregulated in melanoma ICI non-responders.